marine drugs

Article

A Study of 11-[3H]-Tetrodotoxin Absorption,Distribution, Metabolism and Excretion (ADME) inAdult Sprague-Dawley Rats

Bihong Hong 1,2, Hui Chen 2, Jiacai Han 3, Quanling Xie 2, Jianlin He 2, Kaikai Bai 2,Yanming Dong 1,* and Ruizao Yi 2,*

1 Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005,China; bhhong@tio.org.cn

2 Engineering Research Center of Marine Biological Resource Comprehensive Utilization, Third Instituteof Oceanography, State Oceanic Administration, Xiamen 361005, China; chenhui@tio.org.cn (H.C.);qlxie@tio.org.cn (Q.X.); jlhe@tio.org.cn (J.H.); kkbai@tio.org.cn (K.B.)

3 Department of Inspection and Quarantine of Goods, Pingtan Entry-Exit Inspection & Quarantine Bureauof P.R.C, Pingtan 350400, China; ytt629n@163.com

* Correspondences: ymdong@xmu.edu.cn (Y.D.); rzyi@tio.org.cn (R.Y.);Tel.: +86-592-218-5106 (Y.D.); +86-592-219-5265 (R.Y.); Fax: +86-592-219-5265 (R.Y.)

Academic Editor: Andrew TurnerReceived: 23 April 2017; Accepted: 25 May 2017; Published: 2 June 2017

Abstract: Tetrodotoxin (TTX) is a powerful sodium channel blocker that in low doses can safelyrelieve severe pain. Studying the absorption, distribution, metabolism and excretion (ADME) ofTTX is challenging given the extremely low lethal dose. We conducted radiolabeled ADME studiesin Sprague-Dawley rats. After a single dose of 6 µg/(16 µCi/kg) 11-[3H]TTX, pharmacokineticsof plasma total radioactivity were similar in male and female rats. Maximum radioactivity(5.56 ng Eq./mL) was reached in 10 min. [3H]TTX was below detection in plasma after 24 h.The area under the curve from 0 to 8 h was 5.89 h·ng Eq./mL; mean residence time was 1.62 hand t 1

2was 2.31 h. Bile secretion accounted for 0.43% and approximately 51% of the dose was

recovered in the urine, the predominant route of elimination. Approximately 69% was recovered,suggesting that hydrogen tritium exchange in rats produced tritiated water excreted in breath andsaliva. Average total radioactivity in the stomach, lungs, kidney and intestines was higher thanplasma concentrations. Metabolite analysis of plasma, urine and feces samples demonstrated oxidizedTTX, the only identified metabolite. In conclusion, TTX was rapidly absorbed and excreted in rats,a standard preclinical model used to guide the design of clinical trials.

Keywords: tetrodotoxin; ADME; TTX; 11-[3H]TTX; non-clinical pharmacokinetics

1. Introduction

Tetrodotoxin (TTX) is a potent neurotoxin found in a variety of marine and terrestrial species [1–8].Clinical studies suggest that low doses of TTX can safely relieve severe cancer-related pain [9–11]and reduce cue-induced drug craving and anxiety in abstinent heroin addicts [12]. TTX is apowerful sodium channel blocker [13] and the median lethal dose (LD50) is about 10 µg/kg for adultSprague-Dawley (SD) rats with intramuscular injection. After administration, the amount of TTX inthe blood, urine and tissues is extremely low, as well as its metabolites. Both are very polar compounds,which may cause the problem of ion suppression in mass spectrometry (MS) analysis. Therefore, a verysensitive method is required. In recent years, liquid chromatography-mass spectrometry (LC-MS) andespecially LC-MS/MS are regarded as the most popular and sensitive methods for determination ofTTX in plasma and urine [14]. Jen et al. [15] used solid-phase extraction combined with LC-MS/MS

Mar. Drugs 2017, 15, 159; doi:10.3390/md15060159 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2017, 15, 159 2 of 14

to analyze trace amounts of TTX in patients’ serum. The observed limit of detection (LOD) and thelimit of quantification (LOQ) were 0.1 and 1 ng/mL in serum, respectively. Tsai et al. [16] developed anew protocol to determine TTX in the urine and blood of human subjects. The samples were cleansedusing a C18 Sep-Pak cartridge column and determined by LC-MS. The LOD was 15.6 nM (5.0 ng/mL).Fong et al. [17] developed a method that combined double solid phase extraction (C18 and hydrophilicinteraction liquid chromatography) and analyzed by LC-MS/MS. The LOD and LOQ were 0.13 and2.5 ng/mL, respectively. Saito et al. [18] developed a validated method to determine the concentrationof TTX by using MonoSpin CBA or amide column, with the help of LC-MS/MS analysis. The LOQ forthe TTX in serum and urine were 1 and 0.5 ng/mL, respectively. Tsujimura et al. [19] separated andidentified TTX of human serum by the combination of liquid chromatography (LC) equipped with amulti-mode ODS column and tandem mass spectrometry. The LOQ was extremely low as 0.5 ng/mLin serum. Studying the absorption, distribution, metabolism and excretion (ADME) of TTX in adultSD rats is much more challenging based on the best reported LODs for TTX to date.

Using radiolabeled drugs to study ADME has the advantages of high sensitivity because of lowbiological background. Watabe et al. [20] used the low specific radioactivity of [3H]TTX to investigatethe anatomical locations of TTX in pufferfish. After intraperitoneal injection into the pufferfish,[3H]TTX accumulated in most tissues; the highest levels were observed in the skin followed by theliver, intestines and muscle. With time, the radioactivity levels decreased remarkably in most tissuesexcept for the skin and gallbladder. Radioactivity measured in tissues in this study did not necessarilyreflect the true TTX levels because TTX may be metabolized in the pufferfish. Liu et al. [21] usedan isotopic tracer method to study the metabolic dynamics and distribution of TTX in mice afteradministration of [125I]TTX intravenously. The content of [125I]TTX reached the highest concentrationin the blood and lungs after 0.17 h. With time, the content increased in the liver and reached its highestconcentration at 24 h. TTX metabolites were present mainly in the liver. These methods providea reference for the study of pharmacokinetics of TTX, but it does not meet the requirements of theguidelines for the study of non-clinical pharmacokinetics of drugs. We wanted to explore the ADMEof TTX using a standard model, the Sprague-Dawley rat. The objectives of the present study wereto determine the pharmacokinetics (PK) of TTX in SD rats; to identify and quantify metabolites inplasma and excreta; and to determine the rates and routes of excretion of [3H]TTX-related radioactivity,including mass balance in excreta.

2. Results

2.1. Observation of Experimental Animal Health Status

No paradoxical reactions were observed during the experiments after a single intramuscular doseof 6 µg/(16 µCi/kg) 11-[3H]TTX in male or female rats. Thus, this dose of [3H]TTX was well toleratedin SD rats.

2.2. Stability of [3H]TTX

The stability of 11-[3H]TTX was studied before and after administration. As shown in Table 1,the radiochemical purity of 11-[3H]TTX remained about 90% both pre-dose and post-dose, with aaverage purity of 90.48%, while it took only four hours on the TTX administration. It suggested thestability of 11-[3H]TTX was guaranteed during TTX administration.

Table 1. Stability of 11-[3H]TTX.

Dose Solution Name Time Points Radiochemical Purity (%)

11-[3H]TTXPre-dose A (0 h) 90.17Post-dose B (4 h) 90.80

Average 90.48

Mar. Drugs 2017, 15, 159 3 of 14

2.3. Pharmacokinetics of Radioactivity and TTX

Given the hydrogen-tritium exchange of 11-[3H]TTX in the plasma and that formed tritiated waterwould interfere with the results of analysis, plasma samples were dried to remove tritiated water.The plasma drying loss rate reached 65% after 4 h and 100% after 24 h (Table 2). Total radioactivityin plasma was analyzed before and after drying. The results showed that the total radioactivityof tritiated water in plasma increased gradually with the prolonging of time after administration(Figure 1). Pharmacokinetics (PK) parameters using the total radioactivity in dried plasma werecalculated. A summary of the PK parameters of total radioactivity in the plasma is presented in Table 3.After a single intramuscular dose of 6 µg/(16 µCi/kg) 11-[3H]TTX, the PK characteristics of plasmatotal radioactivity in male and female rats were similar. The maximum radioactivity Cmax was reachedafter 10 min; mean Cmax was 5.56 ng Eq./mL. The elimination rapidly peaked and radioactivity in theplasma was unable to be detected 24 h after administration. The area under the curve from 0 to 8 h(AUC0–8 h) was 5.89 h·ng Eq./mL, The mean residence time (MRT) was 1.62 h and t 1

2was 2.31 h.

Table 2. Drying loss rate in plasma (%).

Time Drying Loss Rate (%)

0 NO2 min 13.71 ± 10.045 min 12.98 ± 4.60

10 min 7.45 ± 4.9530 min 29.16 ± 3.48

1 h 38.65 ± 5.402 h 36.90 ± 5.544 h 64.85 ± 7.928 h 75.28 ± 5.37

24 h 100.00 ± 0.0048 h 100.00 ± 0.00

Loss drying rate (%) = (Concentration before drying − Concentration after drying)/Concentration before drying × 100%.Values are presented as mean ± SD. SD, standard deviation (for three male and three female rats).

Table 3. Pharmacokinetic parameters of total radioactivity in dried plasma.

ParameterTotal Radioactivity in Dried Plasma

Male Female Average

Tmax (h) 0.11 ± 0.05 0.11 ± 0.04 0.11 ± 0.04Cmax

a (ng Eq./mL) 5.08 ± 0.40 6.05 ± 1.78 5.56 ± 1.27AUC0–t

a (h·ng Eq./mL) 5.50 ± 0.54 6.28 ± 0.17 5.89 ± 0.56AUC0–∞

a (h·ng Eq./mL) 5.77 ± 0.55 6.83 ± 0.21 6.30 ± 0.69MRT0–t (h) 1.55 ± 0.09 1.68 ± 0.14 1.62 ± 0.12

t 12

(h) 2.03 ± 0.14 2.59 ± 0.32 2.31 ± 0.38a In the calculation Cmax and AUC, 1 g plasma according to 1 mL calculation. Values are presented as mean ± SD.SD, standard deviation (for three male and three female rats).

Mar. Drugs 2017, 15, 159 4 of 14Mar. Drugs 2017, 15, 159  4 of 14 

 

Figure 1. Total radioactivity and time curve of plasma samples (mean of three male and three female rats). 

2.4. Excretion of [3H]TTX in Urine and Feces 

After a single intramuscular dose of 6 μg/(16 μCi/kg) 11‐[3H]TTX  in male and female rats, the 

cumulative recovery rates of radioactivity in urine, feces, cage wash and carcass are summarized in 

Table 4. The mean recovery rate of total radioactivity was 69.35% between 0 and 72 h, and 51.16% 

was recovered  in the urine, 3.87%  in the feces, 10.01%  in the cage wash, and 4.31%  in the carcass. 

After 72 h, the radioactivity of all samples was lower than the limit of quantification. 

Meanwhile, the urine radioactivity before and after drying was calculated. Drying loss rates in 

urine are  summarized  in Table 5. Urine  samples all  contained a  large amount of  tritiated water, 

which increased gradually with the prolonging of time after administration. 

Table 4. Cumulative recovery rate of radioactivity (%) (0–72 h). 

Samples  Over Time (h) Recovery Rate (%)

Urine 

0–8  46.69 ± 13.75 

0–24  50.01 ± 13.34 

0–48  51.09 ± 13.20 

0–72  51.16 ± 13.04 

Feces 

0–24  2.81 ± 1.59 

0–48  3.87 ± 1.88 

0–72  3.87 ± 1.88 

Cage wash 

0–24  9.55 ± 9.47 

0–48  9.82 ± 9.56 

0–72  10.01 ± 9.78 

Carcass  72  4.31 ± 0.80 

Values are presented as mean ± SD. SD, standard deviation (for three male and three female rats). 

Table 5. Drying loss rates in urine (0–72 h). 

Over Time Point (h) Drying Loss Rate (%)

0–8  31.01 ± 8.05 

8–24  41.83 ± 19.78 

24–48  85.75 ± 34.92 

48–72  100.00 ± 0.00 

Loss drying  rate  (%)  =  (Concentration before drying  − Concentration  after drying)/Concentration 

before drying × 100%. Values are presented as mean ± SD. SD, standard deviation (for three male and 

three female rats). 

2.5. Excretion of [3H]TTX in Bile 

After  a  single  intramuscular dose  of  6  μg/(16  μCi/kg)  11‐[3H]TTX  in male  and  female with 

biliary duct cannulation (BDC) rats, cumulative recovery rates of radioactivity  in bile, urine, feces 

Figure 1. Total radioactivity and time curve of plasma samples (mean of three male and three female rats).

2.4. Excretion of [3H]TTX in Urine and Feces

After a single intramuscular dose of 6 µg/(16 µCi/kg) 11-[3H]TTX in male and female rats,the cumulative recovery rates of radioactivity in urine, feces, cage wash and carcass are summarizedin Table 4. The mean recovery rate of total radioactivity was 69.35% between 0 and 72 h, and 51.16%was recovered in the urine, 3.87% in the feces, 10.01% in the cage wash, and 4.31% in the carcass. After72 h, the radioactivity of all samples was lower than the limit of quantification.

Meanwhile, the urine radioactivity before and after drying was calculated. Drying loss rates inurine are summarized in Table 5. Urine samples all contained a large amount of tritiated water, whichincreased gradually with the prolonging of time after administration.

Table 4. Cumulative recovery rate of radioactivity (%) (0–72 h).

Samples Over Time (h) Recovery Rate (%)

Urine

0–8 46.69 ± 13.750–24 50.01 ± 13.340–48 51.09 ± 13.200–72 51.16 ± 13.04

Feces0–24 2.81 ± 1.590–48 3.87 ± 1.880–72 3.87 ± 1.88

Cage wash0–24 9.55 ± 9.470–48 9.82 ± 9.560–72 10.01 ± 9.78

Carcass 72 4.31 ± 0.80

Values are presented as mean ± SD. SD, standard deviation (for three male and three female rats).

Table 5. Drying loss rates in urine (0–72 h).

Over Time Point (h) Drying Loss Rate (%)

0–8 31.01 ± 8.058–24 41.83 ± 19.7824–48 85.75 ± 34.9248–72 100.00 ± 0.00

Loss drying rate (%) = (Concentration before drying − Concentration after drying)/Concentration before drying × 100%.Values are presented as mean ± SD. SD, standard deviation (for three male and three female rats).

Mar. Drugs 2017, 15, 159 5 of 14

2.5. Excretion of [3H]TTX in Bile

After a single intramuscular dose of 6 µg/(16 µCi/kg) 11-[3H]TTX in male and female with biliaryduct cannulation (BDC) rats, cumulative recovery rates of radioactivity in bile, urine, feces and cagewash are summarized in Table 6. The mean recovery rate of total radioactivity was 65.58% between 0and 72 h, except for the carcass; 0.43% was recovered in the bile, 57.26% in the urine, 0.94% in the feces,6.94% in the cage wash, and 4.31% in the carcass.

Table 6. Cumulative recovery rates (%, SD-BDC rats; 0–72 h).

Samples Over Time (h) Recovery Rate (%)

Bile

0–4 0.29 ± 0.080–8 0.42 ± 0.09

0–24 0.43 ± 0.080–48 0.43 ± 0.080–72 0.43 ± 0.08

Urine

0–8 47.98 ± 11.100–24 55.76 ± 13.390–48 57.04 ± 13.360–72 57.26 ± 13.19

Feces0–24 0.47 ± 0.720–48 0.94 ± 1.020–72 0.94 ± 1.02

Cage wash0–24 5.10 ± 2.230–48 6.24 ± 2.650–72 6.94 ± 2.82

Values are presented as mean ± SD. SD, standard deviation (for three male and three female rats).

2.6. Total Radioactivity in Tissues

After a single intramuscular dose of 6 µg/(16 µCi/kg) 11-[3H]TTX in male and female rats,the mean total radioactivity concentrations distributed in whole blood, plasma and tissue-time areshown in Figure 2. The distribution concentrations of testis epididymis and whole brain at timepoints of 0.5, 4, 24 and 72 h were lower than the limit of quantification. The highest concentration ofthe total radioactivity in the other tissues was the highest at the first sampling point (0.5 h), exceptTmax for the intestinal wall, which was at 4 h. Total radioactivity was mainly distributed in thegastrointestinal tract wall after intramuscular administration at 0.5 h. The average total radioactivityin the stomach wall, lung, kidney, heart and intestinal wall were higher than in or near the pointof time in plasma concentrations (3.97 ng Eq./g), 2.54, 2.17, 1.99, 1.59 and 0.924 times the plasmaaverage total radioactivity, respectively. The total radioactivity in the other tissues was lower than theplasma concentrations, and the ovary, spleen, skin, skeletal muscle, liver, thymus and fat were fromhighest to lowest in the order of concentration. After 4 h, the total radioactivity of the intestinal wallreached Cmax, while Cmax of the other tissues decreased. The average total radioactivity of the gastricintestinal wall, lung, kidney, heart, spleen and skin was higher than that time point of the plasmaconcentration (0.862 ng Eq./g), 5.37, 5.36, 4.03, 2.33, 2.33, 1.68 and 1.45 times of the plasma averagetotal radioactivity, respectively. The total radioactivity of skeletal muscle, thymus and fat was lowerthan that of plasma, and the distribution of the liver and ovary uterus was lower than the lower limitof quantification. After 24 h, the tissue total radioactivity was significantly decreased. Only a smallamount of radioactivity was detected in the kidney, gastric, lung, thymus, skeletal muscle and skin.At the last acquisition time point (72 h), the radioactivity of all detected tissues was lower than that ofthe lower limit of quantification.

Mar. Drugs 2017, 15, 159 6 of 14Mar. Drugs 2017, 15, 159  6 of 14 

 

Figure 2. Distribution of [3H]TTX among blood, plasma and tissues in rats following intramuscular 

administration of [3H]TTX at different time points. 

2.7. Metabolite Identification and Profiling 

The LC radiochromatogram of the 11‐[3H]TTX standard is shown in Figure 3. 

 

Figure 3. LC radiochromatogram of 11‐[3H]TTX standard. 

2.7.1. Plasma 

The mixed plasma radioactive metabolite spectrum of the male and female rats at time points of 

0.083 and 4 h is shown in Figure 4. The radioactive metabolite ratio in total radioactivity (%HPLC) is 

shown in Table 7. After a single intramuscular dose of about 6 μg/(16 μCi/kg) 11‐[3H]TTX, prototype 

drug (TTX) was detected in the mixed plasma accounting for total plasma exposure at time points of 

0.083 h  (female, 17.9%; male, 18.8%), 0.5 h  (female, 25.9%; male 22.3%), 1 h  (female, 18.2%; male, 

21.0%), 2 h (female, 24.1%; male, 17.9%) and 4 h (female, 24.8%; male, 32.1%). 

Table 7. The contribution of TTX and metabolic No. 1 (M1) to the total [3H] radioactivity (100%) in 

the plasma of rats at different time points (%). 

3H Resource 0.083 h  0.5 h 1 h 2 h 4 h 

Female Male  Female Male Female Male Female Male  Female  Male

TTX  17.9  18.8  25.9  22.3  18.2  21.0  24.1  17.9  24.8  32.1 

M1  1.80  4.10  4.94  5.75  5.51  3.35  3.35  1.80  4.81  4.26 

Note: The percentages  in Table 7 refer to the ratio of compound’s [3H] signal  intensity to the total 

intensity of [3H] radioactivity LC radiochromatogram, measured by peak area normalization method. 

Figure 2. Distribution of [3H]TTX among blood, plasma and tissues in rats following intramuscularadministration of [3H]TTX at different time points.

2.7. Metabolite Identification and Profiling

The LC radiochromatogram of the 11-[3H]TTX standard is shown in Figure 3.

Mar. Drugs 2017, 15, 159  6 of 14 

 

Figure 2. Distribution of [3H]TTX among blood, plasma and tissues in rats following intramuscular 

administration of [3H]TTX at different time points. 

2.7. Metabolite Identification and Profiling 

The LC radiochromatogram of the 11‐[3H]TTX standard is shown in Figure 3. 

 

Figure 3. LC radiochromatogram of 11‐[3H]TTX standard. 

2.7.1. Plasma 

The mixed plasma radioactive metabolite spectrum of the male and female rats at time points of 

0.083 and 4 h is shown in Figure 4. The radioactive metabolite ratio in total radioactivity (%HPLC) is 

shown in Table 7. After a single intramuscular dose of about 6 μg/(16 μCi/kg) 11‐[3H]TTX, prototype 

drug (TTX) was detected in the mixed plasma accounting for total plasma exposure at time points of 

0.083 h  (female, 17.9%; male, 18.8%), 0.5 h  (female, 25.9%; male 22.3%), 1 h  (female, 18.2%; male, 

21.0%), 2 h (female, 24.1%; male, 17.9%) and 4 h (female, 24.8%; male, 32.1%). 

Table 7. The contribution of TTX and metabolic No. 1 (M1) to the total [3H] radioactivity (100%) in 

the plasma of rats at different time points (%). 

3H Resource 0.083 h  0.5 h 1 h 2 h 4 h 

Female Male  Female Male Female Male Female Male  Female  Male

TTX  17.9  18.8  25.9  22.3  18.2  21.0  24.1  17.9  24.8  32.1 

M1  1.80  4.10  4.94  5.75  5.51  3.35  3.35  1.80  4.81  4.26 

Note: The percentages  in Table 7 refer to the ratio of compound’s [3H] signal  intensity to the total 

intensity of [3H] radioactivity LC radiochromatogram, measured by peak area normalization method. 

Figure 3. LC radiochromatogram of 11-[3H]TTX standard.

2.7.1. Plasma

The mixed plasma radioactive metabolite spectrum of the male and female rats at time points of0.083 and 4 h is shown in Figure 4. The radioactive metabolite ratio in total radioactivity (%HPLC) isshown in Table 7. After a single intramuscular dose of about 6 µg/(16 µCi/kg) 11-[3H]TTX, prototypedrug (TTX) was detected in the mixed plasma accounting for total plasma exposure at time pointsof 0.083 h (female, 17.9%; male, 18.8%), 0.5 h (female, 25.9%; male 22.3%), 1 h (female, 18.2%; male,21.0%), 2 h (female, 24.1%; male, 17.9%) and 4 h (female, 24.8%; male, 32.1%).

Table 7. The contribution of TTX and metabolic No. 1 (M1) to the total [3H] radioactivity (100%) in theplasma of rats at different time points (%).

3H Resource0.083 h 0.5 h 1 h 2 h 4 h

Female Male Female Male Female Male Female Male Female Male

TTX 17.9 18.8 25.9 22.3 18.2 21.0 24.1 17.9 24.8 32.1M1 1.80 4.10 4.94 5.75 5.51 3.35 3.35 1.80 4.81 4.26

Note: The percentages in Table 7 refer to the ratio of compound’s [3H] signal intensity to the total intensity of [3H]radioactivity LC radiochromatogram, measured by peak area normalization method.

Mar. Drugs 2017, 15, 159 7 of 14Mar. Drugs 2017, 15, 159  7 of 14 

 

Figure 4. LC radiochromatogram of mixed plasma at: 0.083 h (a); and 4 h (b). 

2.7.2. Urine and Feces 

Mixed  urine/feces  radioactive metabolite  spectra  of  the male  and  female  rats  between  0–8 

h/0–24  h  are  shown  in  Figures  5  and  6.  The  radioactive metabolites  ratio  in  total  radioactivity 

(%HPLC)  is  shown  in  Table  8.  After  a  single  intramuscular  dose  of  about  6  μg/(16  μCi/kg) 

11‐[3H]TTX, the total radioactivity from the 0–8 h urine was 40.74% (female) and 52.64% (male), the 

prototype drug  (TTX)  accounted  for  26.35%  (female)  and  26.11%  (male),  and M1  (monooxidized 

TTX) accounted  for 7.15%  (female) and 9.43%  (male). Other metabolites were not  identified,  thus 

demonstrating  that  urine  excretion  is  the  predominant  route  of  excretion  of  TTX.  The  total 

radioactivity from the 0–24 h feces was 2.65% (female) and 2.97% (male), the prototype drug (TTX) 

accounted  for  28.19%  (female)  and  12.38%  (male),  and M1  (a  single  oxidation  product  of  TTX) 

accounted for 10.73% (female) and 12.16% (male). Other metabolites were not identified. 

 

Figure 5. LC radiochromatogram of 0–8 h mixed urine for: female (a); and male (b) rats. 

Figure 4. LC radiochromatogram of mixed plasma at: 0.083 h (a); and 4 h (b).

2.7.2. Urine and Feces

Mixed urine/feces radioactive metabolite spectra of the male and female rats between 0–8 h/0–24 hare shown in Figures 5 and 6. The radioactive metabolites ratio in total radioactivity (%HPLC) isshown in Table 8. After a single intramuscular dose of about 6 µg/(16 µCi/kg) 11-[3H]TTX, the totalradioactivity from the 0–8 h urine was 40.74% (female) and 52.64% (male), the prototype drug (TTX)accounted for 26.35% (female) and 26.11% (male), and M1 (monooxidized TTX) accounted for 7.15%(female) and 9.43% (male). Other metabolites were not identified, thus demonstrating that urineexcretion is the predominant route of excretion of TTX. The total radioactivity from the 0–24 h feceswas 2.65% (female) and 2.97% (male), the prototype drug (TTX) accounted for 28.19% (female) and12.38% (male), and M1 (a single oxidation product of TTX) accounted for 10.73% (female) and 12.16%(male). Other metabolites were not identified.

Mar. Drugs 2017, 15, 159  7 of 14 

 

Figure 4. LC radiochromatogram of mixed plasma at: 0.083 h (a); and 4 h (b). 

2.7.2. Urine and Feces 

Mixed  urine/feces  radioactive metabolite  spectra  of  the male  and  female  rats  between  0–8 

h/0–24  h  are  shown  in  Figures  5  and  6.  The  radioactive metabolites  ratio  in  total  radioactivity 

(%HPLC)  is  shown  in  Table  8.  After  a  single  intramuscular  dose  of  about  6  μg/(16  μCi/kg) 

11‐[3H]TTX, the total radioactivity from the 0–8 h urine was 40.74% (female) and 52.64% (male), the 

prototype drug  (TTX)  accounted  for  26.35%  (female)  and  26.11%  (male),  and M1  (monooxidized 

TTX) accounted  for 7.15%  (female) and 9.43%  (male). Other metabolites were not  identified,  thus 

demonstrating  that  urine  excretion  is  the  predominant  route  of  excretion  of  TTX.  The  total 

radioactivity from the 0–24 h feces was 2.65% (female) and 2.97% (male), the prototype drug (TTX) 

accounted  for  28.19%  (female)  and  12.38%  (male),  and M1  (a  single  oxidation  product  of  TTX) 

accounted for 10.73% (female) and 12.16% (male). Other metabolites were not identified. 

 

Figure 5. LC radiochromatogram of 0–8 h mixed urine for: female (a); and male (b) rats. Figure 5. LC radiochromatogram of 0–8 h mixed urine for: female (a); and male (b) rats.

Mar. Drugs 2017, 15, 159 8 of 14Mar. Drugs 2017, 15, 159  8 of 14 

 

Figure 6. LC radiochromatogram of 0–24 h mixed feces for: female (a); and male (b) rats. 

Table 8. The contribution of TTX and metabolic No. 1 (M1) to the total [3H] radioactivity (100%) in 

the urine and feces of rats at different time points (%). 

3H‐Resources Urine (0–8 h) Feces (0–24 h)

Female Male Female Male

TTX  26.35  26.11  28.19  12.38 

M1  7.15  9.43  10.73  12.16 

Note: The percentage  in Table 8 refers to the ratio of compound’s [3H] signal  intensity to the total 

intensity of [3H] radioactivity in the LC radiochromatogram, measured by peak area normalization 

method. 

2.7.3. Identified Metabolites by LC‐MS/MS 

Metabolites were identified from urine and feces of male and female rats by LC‐MS/MS. Except 

for the prototype drug (TTX), only one metabolite was identified, M1 (a single oxidation product of 

TTX). The main metabolites  are  shown  in Figure  7. The  radioactive peak  at  about  10.4 min was 

identified as TTX due to the following results of MS/MS: [M + H]+ m/z 320.1098 (calcd. 320.1088), [M + 

H − H2O]+ m/z 302 and [M + H − 2H2O]+ m/z 284. M1 as the metabolite of TTX, appeared at around 9.2 

min, was oxidized TTX  identified by analyzing the MS/MS results: [M1 + H]+ m/z 336.1054 (calcd. 

336.1043), [M1 + H − H2O]+ m/z 318 and [M1 + H − 2H2O‐O]+ m/z 286. 

NH

NH

OO

HO OHHO

NH

HO

OH

OH

 TTX   

Exact Mass 320.1088 

Figure 6. LC radiochromatogram of 0–24 h mixed feces for: female (a); and male (b) rats.

Table 8. The contribution of TTX and metabolic No. 1 (M1) to the total [3H] radioactivity (100%) in theurine and feces of rats at different time points (%).

3H-ResourcesUrine (0–8 h) Feces (0–24 h)

Female Male Female Male

TTX 26.35 26.11 28.19 12.38M1 7.15 9.43 10.73 12.16

Note: The percentage in Table 8 refers to the ratio of compound’s [3H] signal intensity to the total intensity of [3H]radioactivity in the LC radiochromatogram, measured by peak area normalization method.

2.7.3. Identified Metabolites by LC-MS/MS

Metabolites were identified from urine and feces of male and female rats by LC-MS/MS. Exceptfor the prototype drug (TTX), only one metabolite was identified, M1 (a single oxidation productof TTX). The main metabolites are shown in Figure 7. The radioactive peak at about 10.4 min wasidentified as TTX due to the following results of MS/MS: [M + H]+ m/z 320.1098 (calcd. 320.1088),[M + H − H2O]+ m/z 302 and [M + H − 2H2O]+ m/z 284. M1 as the metabolite of TTX, appeared ataround 9.2 min, was oxidized TTX identified by analyzing the MS/MS results: [M1 + H]+ m/z 336.1054(calcd. 336.1043), [M1 + H − H2O]+ m/z 318 and [M1 + H − 2H2O-O]+ m/z 286.

Mar. Drugs 2017, 15, 159 9 of 14

Mar. Drugs 2017, 15, 159  8 of 14 

 

Figure 6. LC radiochromatogram of 0–24 h mixed feces for: female (a); and male (b) rats. 

Table 8. The contribution of TTX and metabolic No. 1 (M1) to the total [3H] radioactivity (100%) in 

the urine and feces of rats at different time points (%). 

3H‐Resources Urine (0–8 h) Feces (0–24 h)

Female Male Female Male

TTX  26.35  26.11  28.19  12.38 

M1  7.15  9.43  10.73  12.16 

Note: The percentage  in Table 8 refers to the ratio of compound’s [3H] signal  intensity to the total 

intensity of [3H] radioactivity in the LC radiochromatogram, measured by peak area normalization 

method. 

2.7.3. Identified Metabolites by LC‐MS/MS 

Metabolites were identified from urine and feces of male and female rats by LC‐MS/MS. Except 

for the prototype drug (TTX), only one metabolite was identified, M1 (a single oxidation product of 

TTX). The main metabolites  are  shown  in Figure  7. The  radioactive peak  at  about  10.4 min was 

identified as TTX due to the following results of MS/MS: [M + H]+ m/z 320.1098 (calcd. 320.1088), [M + 

H − H2O]+ m/z 302 and [M + H − 2H2O]+ m/z 284. M1 as the metabolite of TTX, appeared at around 9.2 

min, was oxidized TTX  identified by analyzing the MS/MS results: [M1 + H]+ m/z 336.1054 (calcd. 

336.1043), [M1 + H − H2O]+ m/z 318 and [M1 + H − 2H2O‐O]+ m/z 286. 

NH

NH

OO

HO OHHO

NH

HO

OH

OH

 TTX   

Exact Mass 320.1088 Mar. Drugs 2017, 15, 159  9 of 14 

NH

NH

OO

HO OHHO

NH

HO

OH

OHOH

Oxidation TTX   

Exact Mass 336.1043 

Figure  7. Mass‐spectrometric  identification  of  TTX metabolites  in  urine  following  intramuscular 

administration of [3H]TTX. 

3. Discussion 

TTX is a powerful neurotoxin attracting worldwide attention due to its strong sodium channel 

blocker  activities. However,  the  reports  focusing on ADME properties of TTX were  rather poor. 

Only pufferfish  [20,22–24]  and mice  [21] have been used  as  the model  animals  to  systematically 

study the ADME of TTX. What is worse is the two models were improper for forecasing the ADME 

of TTX in human body. 

The rat ADME model is widely used to predict metabolic products of drugs in humans. In this 

study,  rat was utilized  as  the model  animal  for  the  first  time  to  study  the ADME of TTX. After 

intramuscular administration of 11‐[3H]TTX, the [3H] signal could be found in most tissues, and its 

level remained high in some tissues such as intestine, stomach and  lungs. This was  in accordance 

with the previous report by Watabe [20]. The maximum radioactivity Cmax in plasma was reached at 

10 min after the administration of TTX. After that, the radio signal became weak rapidly and it was 

unable to be detected at 24 h after administration (Figure 2). The profile of TTX in the plasma of rats 

was consistent with that studies reported by Liu [21]. Besides, both TTX and the oxidized TTX as the 

metabolite of TTX, were found in the plasma, urine and feces of rats. Moreover, the majority of TTX 

was detected in the urine while only 0.43% of the radioactive dose was excreted in bile, suggesting 

urine  excretion  is  the  predominant  route  of  elimination.  It was  clear  TTX was  excreted  as  the 

prototype drug. 

Some  interesting  results  of  the  study  need deeper discussion.  First,  the distribution  of TTX 

among different tissues was different between rats and other animals. During the study performed 

on  the pufferfish named Fugu rubripes,  the highest  levels of TTX were observed  in  the  intestine, 

followed by the skin, kidney and spleen. The levels decreased remarkably in most tissues except for 

the  skin  and gallbladder  [20]. However, more TTX was observed  in  liver  instead of  skin on  the 

pufferfish  called  Takifugu  rubripes  [22].  Furthermore,  the  preference  of  TTX  distribution  in  some 

tissues  to  that  in plasma was  observed  in  rats. A  surprisingly higher  concentration  of TTX was 

distributed in the stomach, lungs, kidney and heart than in the plasma at the point of 0.5 h. This may 

be  partially  explained  by  the  abundant  blood  or  a  large  amount  of  highly  viscous  material 

distributed  in  those  tissues.  It  is also consistent with other  investigators who have  found  that  the 

highest concentration of intravenous administered [125I]TTX was distributed in the lung at the point 

of 0.5 h [21]. Finally, TTX was apt to be retained in intestines of animals. It had been reported TTX 

might be cumulative  in  the  intestine of pufferfish after an  intraperitoneal  injection administration 

[20].  Similar  results were  observed  in  this  experiment.  The  total  radioactivity  of  [3H]TTX  in  the 

intestine of  rats  reached  its peak at 4 h after  the  treatment while  the  [3H] signals of other  tissues 

decreased.  It may  be  related with  the  amount  of  receptor,  but  it  remains  uncertain why  TTX 

accumulated in the intestines. Based on the analysis above, it suggested the ADME property of TTX 

among animal species may be different and complex. More investigation is needed. 

The  determination  of  TTX  and  its metabolites  is  one  of  technical  barriers  to  be  overcome. 

Although the highest dose of TTX (6 μg/kg) that could be tolerated by rats was given, the dose is 

extremely low in terms of detection. Meanwhile, TTX has extensive metabolism in animals, such as 

4,9‐anhydroTTX, 5,6,11‐trideoxyTTX, 11‐deoxyTTX and other oxidized TTXs [25,26], likely resulting 

in the awful low concentration of TTX metabolites in rats’ plasma, urine and feces. Therefore, most 

Figure 7. Mass-spectrometric identification of TTX metabolites in urine following intramuscularadministration of [3H]TTX.

3. Discussion

TTX is a powerful neurotoxin attracting worldwide attention due to its strong sodium channelblocker activities. However, the reports focusing on ADME properties of TTX were rather poor. Onlypufferfish [20,22–24] and mice [21] have been used as the model animals to systematically study theADME of TTX. What is worse is the two models were improper for forecasing the ADME of TTX inhuman body.

The rat ADME model is widely used to predict metabolic products of drugs in humans. In thisstudy, rat was utilized as the model animal for the first time to study the ADME of TTX. Afterintramuscular administration of 11-[3H]TTX, the [3H] signal could be found in most tissues, and itslevel remained high in some tissues such as intestine, stomach and lungs. This was in accordancewith the previous report by Watabe [20]. The maximum radioactivity Cmax in plasma was reached at10 min after the administration of TTX. After that, the radio signal became weak rapidly and it wasunable to be detected at 24 h after administration (Figure 2). The profile of TTX in the plasma of ratswas consistent with that studies reported by Liu [21]. Besides, both TTX and the oxidized TTX as themetabolite of TTX, were found in the plasma, urine and feces of rats. Moreover, the majority of TTX wasdetected in the urine while only 0.43% of the radioactive dose was excreted in bile, suggesting urineexcretion is the predominant route of elimination. It was clear TTX was excreted as the prototype drug.

Some interesting results of the study need deeper discussion. First, the distribution of TTX amongdifferent tissues was different between rats and other animals. During the study performed on thepufferfish named Fugu rubripes, the highest levels of TTX were observed in the intestine, followed bythe skin, kidney and spleen. The levels decreased remarkably in most tissues except for the skin andgallbladder [20]. However, more TTX was observed in liver instead of skin on the pufferfish calledTakifugu rubripes [22]. Furthermore, the preference of TTX distribution in some tissues to that in plasmawas observed in rats. A surprisingly higher concentration of TTX was distributed in the stomach,lungs, kidney and heart than in the plasma at the point of 0.5 h. This may be partially explained bythe abundant blood or a large amount of highly viscous material distributed in those tissues. It isalso consistent with other investigators who have found that the highest concentration of intravenousadministered [125I]TTX was distributed in the lung at the point of 0.5 h [21]. Finally, TTX was apt tobe retained in intestines of animals. It had been reported TTX might be cumulative in the intestineof pufferfish after an intraperitoneal injection administration [20]. Similar results were observed inthis experiment. The total radioactivity of [3H]TTX in the intestine of rats reached its peak at 4 h after

Mar. Drugs 2017, 15, 159 10 of 14

the treatment while the [3H] signals of other tissues decreased. It may be related with the amount ofreceptor, but it remains uncertain why TTX accumulated in the intestines. Based on the analysis above,it suggested the ADME property of TTX among animal species may be different and complex. Moreinvestigation is needed.

The determination of TTX and its metabolites is one of technical barriers to be overcome. Althoughthe highest dose of TTX (6 µg/kg) that could be tolerated by rats was given, the dose is extremely lowin terms of detection. Meanwhile, TTX has extensive metabolism in animals, such as 4,9-anhydroTTX,5,6,11-trideoxyTTX, 11-deoxyTTX and other oxidized TTXs [25,26], likely resulting in the awful lowconcentration of TTX metabolites in rats’ plasma, urine and feces. Therefore, most of the metabolites inthe matrix could not be identified by LC-MS/MS. In addition to the LC-MS/MS, mass defect filtration(MDF) technique was actually applied by the authors to search the metabolic products of TTX inphase I and II in the urine samples and the results showed no mass peak of TTX related metabolites.Additionally, the identification of TTX metabolites is another challenge. Whether M1 was 11-oxoTTXwas uncertain though these two compounds presented the same exact mass and the similar expectedfragmentation law of MS. M1 might be 11-oxoTTX, which is a compound isolated from pufferfish,because the compound is a natural analog of TTX [27]. Exploring more suitable methods to identifyTTX metabolites is challenging and it will be the subject of ongoing studies.

4. Materials and Methods

4.1. Regents

TTX standard (assay 98.3%) was provided by the Third Institute of Oceanography StateOceanic, Fujian, China. The 11-[3H]TTX (Figure 8, radiochemical purity 90.71%, specific radioactivity9.86 Ci/mmol) was prepared according to the previously published methods [28] with slight modifications.In brief, lyophilized TTX was transformed to 11-oxoTTX by dicyclohexylcarbodiimide (DCC), usingH3PO4 as the catalytic agent. Then, the 11-oxoTTX was deoxidized by tritiated sodium borohydrideand the resultant 11-[3H]TTX was obtained with high purity by HPLC.

Mar. Drugs 2017, 15, 159  10 of 14 

of the metabolites in the matrix could not be identified by LC‐MS/MS. In addition to the LC‐MS/MS, 

mass defect filtration (MDF) technique was actually applied by the authors to search the metabolic 

products of TTX in phase I and II in the urine samples and the results showed no mass peak of TTX 

related  metabolites.  Additionally,  the  identification  of  TTX  metabolites  is  another  challenge. 

Whether M1 was 11‐oxoTTX was uncertain though these two compounds presented the same exact 

mass  and  the  similar  expected  fragmentation  law  of MS. M1 might  be  11‐oxoTTX, which  is  a 

compound  isolated  from  pufferfish,  because  the  compound  is  a  natural  analog  of  TTX  [27]. Exploring more suitable methods to identify TTX metabolites is challenging and it will be the subject 

of ongoing studies. 

4. Materials and Methods 

4.1. Regents 

TTX  standard  (assay  98.3%)  was  provided  by  the  Third  Institute  of  Oceanography  State 

Oceanic, Fujian, China. The 11‐[3H]TTX (Figure 8, radiochemical purity 90.71%, specific radioactivity 

9.86  Ci/mmol)  was  prepared  according  to  the  previously  published  methods  [28]  with  slight 

modifications. In brief, lyophilized TTX was transformed to 11‐oxoTTX by dicyclohexylcarbodiimide 

(DCC), using H3PO4 as the catalytic agent. Then, the 11‐oxoTTX was deoxidized by tritiated sodium 

borohydride and the resultant 11‐[3H]TTX was obtained with high purity by HPLC. 

NH

NH

NH2

HO

O

O

HO

OHOH

OHH

3H

HO

 

Figure 8. The structure of 11‐[3H]TTX. 

4.2. Radiolabeled Study Drug 

The  study drug was  adjusted  to  a  final  specific  radioactivity  of  2842.875 μCi/mg  (6,311,183 

dpm/μg) by dilution with non‐radiolabeled TTX  standard. The dosing  form  concentration was 6 

μg/g, with a radioactivity concentration of 36,519,188 dpm/g. Based on the available LD50 of TTX in 

rats of about 10 μg/kg, the chosen dose of 6 μg/(16 μCi/kg) (TTX + 11‐[3H]TTX) was expected to be 

tolerated. 

4.3. Analysis of the Radiochemical Purity of TTX 

The  radioactive  constituents were  separated  by HPLC  equipped with  an  automatic  fraction 

collector. The HPLC was performed  on  a  Shimadzu UFLC‐20A, using  a Waters Xbridge Amide 

HILIC (Milford, MA, USA) column (4.6 × 100 mm, 3.5 μm) equilibrated with 30% solvent A (0.1% 

formic  acid  aqueous  solution)  and  70%  solvent  B  (acetonitrile,  J&K  Scientific,  Beijing,  China). 

Consequently,  the eluent was collected  into scintillation vials at 30‐s  intervals. The radiochemical 

purity of the sample in each vial was recorded and analyzed by LSC. 

4.4. Animals and Experimental Design 

Sprague‐Dawley (SD) rats (8–10 weeks old at the time of the experiment, 240 ± 50 g in weight) 

were  obtained  from  the  Beijing  Weitong  Lihua  Experimental  Animal  Technology  Co.,  Ltd. 

(Laboratory animal production license No.: SCXK [Beijing] 2012‐0001, Beijing, China). 

Figure 8. The structure of 11-[3H]TTX.

4.2. Radiolabeled Study Drug

The study drug was adjusted to a final specific radioactivity of 2842.875 µCi/mg (6,311,183 dpm/µg)by dilution with non-radiolabeled TTX standard. The dosing form concentration was 6 µg/g, with aradioactivity concentration of 36,519,188 dpm/g. Based on the available LD50 of TTX in rats of about10 µg/kg, the chosen dose of 6 µg/(16 µCi/kg) (TTX + 11-[3H]TTX) was expected to be tolerated.

4.3. Analysis of the Radiochemical Purity of TTX

The radioactive constituents were separated by HPLC equipped with an automatic fractioncollector. The HPLC was performed on a Shimadzu UFLC-20A, using a Waters Xbridge Amide HILIC(Milford, MA, USA) column (4.6 × 100 mm, 3.5 µm) equilibrated with 30% solvent A (0.1% formicacid aqueous solution) and 70% solvent B (acetonitrile, J&K Scientific, Beijing, China). Consequently,the eluent was collected into scintillation vials at 30-s intervals. The radiochemical purity of the samplein each vial was recorded and analyzed by LSC.

Mar. Drugs 2017, 15, 159 11 of 14

4.4. Animals and Experimental Design

Sprague-Dawley (SD) rats (8–10 weeks old at the time of the experiment, 240 ± 50 g in weight)were obtained from the Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Laboratoryanimal production license No.: SCXK [Beijing] 2012-0001, Beijing, China).

4.5. Pharmacokinetic Study

For pharmacokinetic studies, a group of 6 rats, half male and half female, with jugular veincatheterization (JVC), were each administered an intramuscular (i.m.) injection with an average6 µg/(16 µCi/kg) body weight of [3H]TTX. Serial blood samples (~0.3 mL) were collected in heparinizedtubes via the jugular vein before and at time points of 0.033, 0.083, 0.167, 0.5, 1.0, 2.0, 4.0, 8.0, 24.0,and 48.0 h after administration. Plasma was separated and stored frozen at −20 ◦C until analysis.

4.6. Urine and Feces Excretion Study

For urine and feces excretion studies, 6 rats, 3 male and 3 female, were each administered an i.m.injection with an average 6 µg/(16 µCi/kg) body weight of [3H]TTX. Samples of urine, feces and cagewash solution were collected between 0–8 h, 8–24 h (feces only 0–24 h), 24–48 h, and 48–72 h afteradministration. All samples were stored frozen at −20 ◦C until analysis.

4.7. Bile Excretion Study

For bile excretion studies, a group of 6 rats, half male and half female, with biliary duct cannulation(BDC), were each administered an i.m. injection of 6 µg/(16 µCi/kg) body weight of [3H]TTX. Sampleswere collected between 0 and 4 h, 4 and 8 h, 8 and 24 h, 24 and 48 h, and 48 and 72 h after administration.All samples were stored frozen at −20 ◦C until analysis.

4.8. Tissue Distribution Study

For tissue distribution studies, a group of 24 rats (to minimize animal use, tissue samples atthe 72 h time point came from the rats used in the urine and feces excretion studies), half male andhalf female, were each administered an i.m. injection of 6 µg/(16 µCi/kg) body weight of [3H]TTX.Samples of tissue (heart, liver, spleen, lung, kidney, whole brain, skin [neck], fat [abdomen], skeletalmuscle [non-injection leg femur], gastric wall, intestinal wall, thymus, test is epididymis and uterusovary) and carcass were collected at time points of 0.5, 4, 24, and 72 h after administration. All sampleswere stored frozen at −20 ◦C until analysis.

4.9. Control Group

For the control group (a male rat), samples of whole blood, plasma, tissue, and carcass werecollected at the 24 h time point; samples of urine and feces were collected between 0 and 24 h.All samples were stored frozen at −20 ◦C until analysis.

4.10. General Methods

4.10.1. Determination of Total Radioactivity

Total radioactivity in plasma, urine, bile, cage wash, whole blood, tissue and carcass wasdetermined by liquid scintillation counter (LSC) using a Model Tri-Carb 3110TR (Perkin Elmer,Waltham, MC, USA). External standard methods were used for quenching correction, with countsat least 5 min per sample. Set scintillation counting automatically counts per minute (CPM) wereconverted to decays per minute (DPM). The value of the corresponding sample of the control groupwas measured as the background of the instrument, with calibration of samples in the experimentalgroup. The background signal of the instrument was set to zero.

Mar. Drugs 2017, 15, 159 12 of 14

To determine the total radioactivity of plasma and urine samples, two samples were used: one(about 0.05 g) was added to scintillation liquid (5 mL) and determined directly by LSC, while the otherwas dried and purified water (0.1 mL) was added to reconstitute then scintillation liquid (5 mL) wasadded, followed by LSC. Bile and cage wash levels were determined directly by LSC. Feces, wholeblood, tissue and carcass were homogenized in 50% isopropanol, weighed 2 samples (about 0.1 g),then KOH (6 M, 100 µL) was added, heated in a water bath, and then cooled. Scintillation liquid (5 mL)was added after glacial acetic acid and methanol were used to reconstitute, followed by LSC.

4.10.2. Sample Preparation for Metabolite Identification and Profiling

Plasma samples that came from the pharmacokinetic study group were pooled by time points of0.083, 0.5, 1.0, 2.0 and 4.0 h, mixing equal volumes of plasma from male and female rats at each timepoint. Each pooled plasma sample was extracted with 0.1% formic acid-methanol, after which theextracted solution was dried by nitrogen and the mixture of 0.1% HOOC–99.9%CH3OH:H2O (1:1, v/v)was added to reconstitute. Urine samples that came from the urine excretion study group werepooled between 0 and 8.0 h, mixing equal volumes of urine from male and female rats. Each pooledurine sample was precipitated with 0.3% formic acid–methanol, centrifuged, and then separatedinto solution.

Feces samples that came from the feces excretion study group were pooled between 0 and 24.0 h,mixing equal weights of feces from male and female rats. Each pooled feces sample was extractedwith 0.1% formic acid–methanol, after which the extracted solution was dried by nitrogen and themixture of 0.1% HOOC–99.9%CH3OH:H2O (1:1, v/v) was added to reconstitute. The radioactivity ofthe prepared samples was determined by LSC and metabolites were identified by LC-MS/MS.

4.10.3. Pharmacokinetic Analysis

According to the radioactivity of plasma samples, the following main pharmacokineticparameters were analyzed using the non-compartmental pharmacokinetics data analysis softwareWinNonlin (Version 6.3, Pharsight Corp., Los Angeles, CA, USA): maximum concentration (Cmax),time-to-maximum concentration (Tmax), area under the curve from 0 to the time (AUC0–t) and to thelast measurable plasma concentration point (AUC0–∞), half-life (t 1

2), and mean residence time (MRT).

4.10.4. Metabolite Identification and Profiling

Prepared plasma, urine and feces samples were separated by HPLC equipped with an automaticfraction collector to collect eluent at 30-second intervals into scintillation vials, using LSC to analyzeand convert by ARC® Convert software (Los Angeles, CA, USA), to obtain reconstituted radioactivemetabolite profiles. The spectrum of the radioactive metabolites of each sample was treated by integralsto obtain the peak area. The percentage of each metabolite and total radioactivity of each sample wascalculated according to the peak area (%HPLC).

HPLC separation was performed on a Shimadzu UFLC-20A, using a Waters Amide HILIC column(4.6 × 100 mm, 3.5 µm) equilibrated with 20% solvent A (0.1% formic acid aqueous solution) and80% solvent B (acetonitrile) at a flow rate of 1 mL/min. After 3 min, a linear gradient was run to 60%solvent A over 7 min and maintained for 3 min, followed by a linear gradient to 20% solvent A over2 min and maintained for 9 min.

Analysis and identification of metabolites was conducted using a Thermo LTQ Orbitrap XLLC-MS/MS System (Thermo Scientific, Waltham, MA, USA) equipped with electrospray ionization(ESI). Detection was performed in positive ion mode under the following conditions: ion spray voltageat 3500 V, 350 ◦C, skimmer Offset at 10 units, tube lens offset at 80 units, sheath gas pressure at 20 units,auxiliary gas pressure at 10 units, sweep gas flow at 10 units, collision energy (CE) at 35 eV, and scantype at Q1 MS, MS/MS. Xcalibr software (Thermo Scientific, Waltham, MA, USA) was used for systemcontrol and data acquisition.

Mar. Drugs 2017, 15, 159 13 of 14

5. Conclusions

The present study investigated ADME of a single intramuscular dose of [3H]-radiolabeled TTX inadult Sprague-Dawley rats. The dose was rapidly absorbed and distributed mainly in the stomach,lungs, kidney and intestines. It was eliminated quickly and was unable to be detected in the plasma24 h after administration. It also demonstrated that oxidation of TTX is the identified metabolite, exceptprototype drug (TTX). The results provide key reference information for the design and optimizationof clinical trials of TTX.

Acknowledgments: This work was supported financially by the Public Science and Technology Research FundsProjects of Ocean (No. 201405016), Xiamen Southern Oceanographic Center (No. 13GYY001NF05), and Scientificand Technological Innovation Platform of Fujian Province (No. 2014H2006).

Author Contributions: Bihong Hong, Yanming Dong and Ruizao Yi conceived and designed the experiments;Bihong Hong, Hui Chen, Jiacai Han and Quanling Xie carried out the experiments and analyzed the data;Bihong Hong drafted the paper; and Jianlin He and Kaikai Bai analyzed the data and offered advice for theimprovement of language. All authors read and approved the final manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Mahmud, Y.; Tanu, M.B.; Noguchi, T. First occurrence of a food poisoning incident due to ingestion ofTakifugu oblongus, along with a toxicological report on three marine puffer species in Bangladesh. J. Food Hyg.Soc. Jpn. 1999, 40, 473–480. [CrossRef]

2. Sui, L.M.; Chen, K.; Hwang, P.A.; Hwang, D.F. Identification of tetrodotoxin in marine gastropods implicatedin food poisoning. J. Nat. Toxins 2002, 11, 213–220. [PubMed]

3. Jang, J.; Yotsu-Yamashita, M. Distribution of tetrodotoxin, Saxitoxin, and their analogs among tissues of thepuffer fish Fugu pardalis. Toxicon 2006, 48, 980–987. [CrossRef] [PubMed]

4. Huang, J.-H.; Lin, H.-L. Identification and quantification of tetrodotoxin in the marine gastropod Nassarius byLC-MS. Toxicon 2008, 51, 774–779. [CrossRef] [PubMed]

5. Chulanetra, M.; Sookrung, N.; Srimanote, P.; Indrawattana, N.; Thanongsaksrikul, J.; Sakolvaree, Y.;Chongsa-Nguan, M.; Kurazono, H.; Chaicumpa, W. Toxic marine puffer fish in Thailand Seas andtetrodotoxin they contained. Toxins 2011, 3, 1249–1262. [CrossRef] [PubMed]

6. Kim, Y.H.; Brown, G.H.; Mosher, H.S.; Fuhrman, F.A. Tetrodotoxin: Occurrence in Atelopid frogs of CostaRica. Science 1975, 189, 151–152. [CrossRef] [PubMed]

7. Yasumoto, T.; Yotsu, M.; Naoki, H. New tetrodotoxin analogues from the newt Cynops ensicauda. J. Am. Chem. Soc.1988, 110, 2344–2345. [CrossRef]

8. Mebs, D.; Yotsu-Yamashita, M. Tetrodotoxin in North-American newts. Toxicon 2012, 60, 1–120. [CrossRef]9. Hagen, N.A.; Fisher, K.M.; Lapointe, B.; du Souich, P.; Chary, S.; Moulin, D.; Sellers, E.; Ngoc, A.H.; Canadian

Tetrodotoxin Study Group. An open-label, multi-dose efficacy and safety study of intramuscular tetrodotoxinin patients with severe cancer-related pain. J. Pain Symptom Manag. 2007, 34, 171–182. [CrossRef] [PubMed]

10. Hagen, N.A.; du Souich, P.; Lapointe, B.; Ong-Lam, M.; Dubuc, B.; Walde, D.; Love, R.; Ngoc, A.H.; CanadianTetrodotoxin Study Group. Tetrodotoxin for moderate to severe cancer pain: A randomized, double blind,parallel design multicenter study. J. Pain Symptom Manag. 2008, 35, 420–429. [CrossRef] [PubMed]

11. Hagen, N.A.; Lapointe, B.; Ong-Lam, M.; Dubuc, B.; Walde, D.; Gagnon, B.; Love, R.; Goel, R.; Hawley, P.;Ngoc, A.H.; et al. A multicentre open-label safety and efficacy study of tetrodotoxin for cancer pain.Curr. Oncol. 2011, 18, 109–116. [CrossRef]

12. Shi, J.; Liu, T.T.; Wang, X.; David, H.E.; Zhao, L.Y.; Zhang, X.L.; Lu, L. Tetrodotoxin reduces cue-induced drugcraving and anxiety in abstinent heroin addicts. Pharmacol. Biochem. Behav. 2009, 92, 603–607. [CrossRef][PubMed]

13. Catterall, W.A. Structure and function of voltage-gated ion channels. Annu. Rev. Biochem. 1995, 64, 493–531.[CrossRef] [PubMed]

14. Leung, S.Y.; Fong, M.W.; Tsoi, Y.K. Analytical Challenges: Determination of Tetrodotoxin in Human Urineand Plasma by LC-MS/MS. Mar. Drugs 2011, 9, 2291–2303. [CrossRef] [PubMed]

Mar. Drugs 2017, 15, 159 14 of 14

15. Jen, H.-C.; Lin, S.-J.; Tsai, Y.-H.; Chen, C.-H.; Lin, Z.-C.; Hwang, D.-F. Tetrodotoxin poisoning evidenced bysolid-phase extraction combining with liquid chromatography-tandem mass spectrometry. J. Chromatogr. B2008, 871, 95–100. [CrossRef] [PubMed]

16. Tsai, Y.H.; Hwang, D.F.; Cheng, C.A.; Hwang, C.C.; Deng, J.F. Determination of tetrodotoxin in humanurine and blood using C18 cartridge column, ultrafiltration and LC-MS. J. Chromatogr. B 2006, 832, 75–80.[CrossRef] [PubMed]

17. Fong, B.M.-W.; Tam, S.; Tsui, S.-H.; Leung, K.S.-Y. Development and validation of a high-throughput doublesolid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination oftetrodotoxin in human urine and plasma. Talanta 2011, 83, 1030–1036. [CrossRef] [PubMed]

18. Saito, T.; Miura, N.; Namera, A.; Ota, S.; Miyazaki, S.; Inokuchi, S. A Rapid Sample Preparation ProcedureUsing MonoSpin CBA and Amide Columns for Tetrodotoxin Detection in Serum and Urine UsingLC–MS/MS Analysis. Chromatographia 2014, 77, 687–693. [CrossRef]

19. Tsujimura, K.; Yamanouchi, K. A rapid method for tetrodotoxin (TTX) determination by LC-MS/MS fromsmall volumes of human serum, and confirmation of pufferfish poisoning by TTX monitoring. Food Addit.Contam. Part A 2015, 32, 977–983. [CrossRef] [PubMed]

20. Watabe, S.; Sato, Y.; Nakaya, M.; Nogawa, N.; Oohashi, K.; Noguchi, T.; Morikawa, N. Distribution of tritiatedtetrodotoxin administered intraperitoneally to pufferfish. Toxicon 1987, 25, 1283–1289. [CrossRef]

21. Liu, M.; Zhao, C.X.; Qi, X.L.; Yu, M.B.; Lin, F.S. Metabolic dynamics and distribution of tetrodotoxin in mice.Chin. Pharmacol. Bull. 2006, 22, 507–508.

22. Tatsuno, R.; Gao, W.; Ibi, K.; Mine, T.; Okita, K.; Nishihara, G. N.; Takatani, T.; Arakawa, O. Profile differencesin tetrodotoxin transfer to skin and liver in the pufferfish Takifugu rubripes. Toxicon 2017, 130, 73–78. [CrossRef][PubMed]

23. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Pharmacokinetics oftetrodotoxin in puffer fish Takifugu rubripes by a single administration technique. Toxicon 2008, 6, 1051–1059.[CrossRef] [PubMed]

24. Matsumoto, T.; Nagashima, Y.; Kusuhara, H.; Ishizaki, S.; Shimakura, K.; Shiomi, K. Evaluation of hepaticuptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes. Toxicon 2008, 51, 369–374. [CrossRef][PubMed]

25. Yotsu-Yamashita, M.; Jang, J.H.; Cho, Y.; Konoki, K. Optimization of simultaneous analysis of tetrodotoxin,4- epi tetrodotoxin, 4,9-anhydrotetrodotoxin, and 5,6,11-trideoxytetrodotoxin by hydrophilic interactionliquid chromatography–tandem mass spectrometry. Forensic Toxicol. 2011, 29, 61–64. [CrossRef]

26. Kudo, Y.; Yasumoto, T.; Konoki, K.; Cho, Y.; Yotsu-Yamashita, M. Isolation and Structural Determinationof the First 8-epi-type Tetrodotoxin Analogs from the Newt, Cynops ensicauda popei, and Comparison ofTetrodotoxin Analogs Profiles of This Newt and the Puffer Fish, Fugu poecilonotus. Mar. Drugs 2012, 10,655–667. [CrossRef] [PubMed]

27. Khora, S.S.; Yasumoto, T. Isolation of 11-oxotetrodotoxin from the puffer Arothron nigropunctatus. Tetrahedron Lett.1989, 30, 4393–4394. [CrossRef]

28. Wu, B.Q.; Yang, L.; Kao, C.Y.; Levinson, S.R.; Yotsu-yamashita, M.; Yasumoto, T. 11-oxotetrodotoxin and aspecifically labelled 3H-tetrodotoxin. Toxicon 1996, 34, 407–416. [CrossRef]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).